Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is a re-emerging vector-borne infection with an alarming increase in 40% of the reported cases in the United States during the last year in which data is available (2002). This increase in reported cases, coupled with the lack of any current vaccine, makes B. burgdorferi an important public health issue particularly in endemic states where it contributes to significant morbidity. The ability of B. burgdorferi to cause infection in mammals requires rapid adaptation to the mammalian host conditions following transmission from ticks. Genome-wide transcriptional analysis has revealed that a number of genes on linear plasmid 54 (Ip54) exhibit differential expression to either mammalian or tick imposed conditions. The objective of this proposal is to identify genes on Ip54 required for infectivity in the murine model of Lyme disease among the subset that exhibit differential expression to various external signals. The first specific aim is to generate mutants in five specific Ip54-encoded genes by using a customized transposon that will allow for both isolation of mutants in B. burgdorferi following in vitro selection and permit analysis of their infectivity phenotype in mice. The second specific aim is to determine the levels of infectivity of the mutants in the murine model of Lyme disease using C3H/HeN mice. The 50% infectivity dose (ID50) and tissue dissemination characteristics of the five mutants will be determined in comparison with their isogenic parental clones. The final specific aim is to determine the role of selected Ip54 genes in protective immunity in the murine model of Lyme disease and ascertain the pathogenic mechanism(s) mediated by the gene products. These studies will help to characterize borrelial determinants on Ip54 that are critical for infection that may have utility as much needed vaccine candidates to protect against Lyme borreliosis.